It is well known that using the techniques of molecular biology specific sequences may be selected and engineered to modify useful microorganisms to create molecules that may be used for treating cancer, leukemia and HIV-related diseases. Generally, bodily states in mammals, including infectious disease states are directly affected by proteins. Those proteins acting directly or through enzymatic functions contribute in large proportion to many diseases in animals and humans. In the past, therapeutic treatment has focused on interactions with those proteins in an effort to moderate the disease. Attempts have also been made to moderate the actual production of such proteins by interaction with molecules that directly affect their synthesis, i.e., the DNA sequence that underlies the gene product. It is well known that by interfering with the production of the proteins, the effect of the therapeutic results can be maximized. Likewise, therapeutic approaches have been developed that interfere with gene expression, leading to undesired formations which need to be patrolled.
There are numerous methods that have been formulated for inhibiting specific gene expression which have been adopted to some degree and have been defined as antisense nucleic acids. The basic approach is that an oligonucleotide analog complimentary to a specific targeted messenger RNA or mRNA sequence is used. Pertinent references include those of Stein and Cohen (1988); Walder (1988); Marcus-Sekura (1988); Zan (1988); Van der Krol (1988) and Matteucci and Wagner (1996). Each of the foregoing concern general antisense theory and prior techniques.
For example, prior attempts to inhibit HIV gene synthesis by various antisense approaches have been made by a number of researchers. Zamecnik and coworkers have used a transcriptase primer site and splice donor/acceptor sites (P. C. Zamecnik, et al., 1986). Goodchild and coworkers have made phosphodiester compounds targeted to the initiation sites for translation, the cap site, the polyadenylation signal, the 5' repeat region and a site between the gag and pol genes (J. Goodchild, et al., 1988). In the Goodchild study, the greatest activity was achieved by targeting the polyadenylation signal. Agrawal and coworkers have extended the studies of Goodchild by using chemically modified oligonucleotide analogs, which were also targeted to the cap and splice donor/acceptor sites (A. Agrawal, et al., 1988). A portion of one of these oligonucleotide analogs overlapped a portion of the HIV TAR region but was not found to have an exemplary effect. Neither was this oligonucleotide analog designed to interfere with the HIV TAR region. Agrawal and coworkers have used oligonucleotide analogs targeted to the splice donor/acceptor site to inhibit HIV infection in early infected and chronically infected cells (S. Agrawal, et al., 1989).
Sarin and coworkers have also used chemically modified oligonucleotide analogs targeted to the cap and splice donor/acceptor sites (P. S. Sarin, et al., 1988). Zia and coworkers, on the other hand, used an oligonucleotide analog targeted to a splice acceptor site to inhibit HIV (J. A. Zia, et al., 1988). Matsukura and coworkers synthesized oligonucleotide analogs targeted to the initiation of translation of the rev gene mRNA (M. Matsukura, et al., 1987; R. L. Letsinger, et al., 1987; and R. L. Letsinger, et al., 1989). Mori and coworkers used a different oligonucleotide analog targeted to the same region as targeted by Matsukura (K. Mori, et al., 1989). Shibahara and coworkers used oligonucleotide analogs targeted to a splice acceptor site as well as to the reverse transcriptase primer binding site (S. Shibahara, et al., 1989). Letsinger and coworkers synthesized and tested oligonucleotide analogs with conjugated cholesterol targeted to a splice site. Stevenson and Iversen conjugated polylysine to oligonucleotide analogs targeted to the splice donor and the 5'-end of the first exon of the tat gene (Stevenson and Iversen, 1989). Buck and coworkers have recently described the use of phosphate-methylated DNA oligonucleotides targeted to HIV mRNA and DNA (H. M. Buck, et al., 1990). In addition, a U.S. Patent issued to Ecker, has disclosed the use of synthetic oligonucleotides to inhibit the activity of the HIV virus (U.S. Pat. No. 5,166,195). The method utilized, however, lacks in forethought and efficacy.
The need has, therefore, arisen for a systematic method and apparatus for identifying target DNA and RNA sequences in a predictable, logical pattern. Also required is a process that optimizes a ranking of nucleic acid sequences for targeting with antisense oligonucleotides. In addition, there is a long felt need for a means of assessing the thermodynamics and strength of nucleic acid hybridization in sequences that are to be the targets of antisense gene targeting technology in order to efficiently and expeditiously affect their expression.